Report
Electrochemical Detection IN MICROCOLUMN SEPARATIONS
I
t is difficult to find an area of analytical chemistry that has not been affected by the general movement toward the miniaturization of existing techniques; separation science has by no means been excluded from this trend. In particular, major advances toward miniaturization have been made in HPLC and in capillary zone electrophoresis (CZE); thus, it is now possible to analyze samples with volumes in the nanoliter to femtoliter range. With the development of low-volume separations comes the need to reassess
A n d r e w G. Ewing Jody M. M e s a r o s Peter F. Gavin The Pennsylvania State University 0003-2700/94/0366-527A/$04.50/0 © 1994 American Chemical Society
Electrochemical detection is ideally suited for the miniaturization of HPLC and CZE detection strategies to accommodate minute amounts of analyte. Conventional detection methods designed for macroseparations cannot effectively be applied to extremely low volumes; therefore, the development of detection schemes for microseparations is challenging. The focus of this Report is on electrochemical detection schemes that have been used
successfully for microseparations resulting from the miniaturization of HPLC and CZE. In addition, an example of the utility of microseparations using electrochemical detection—single-cell analysis—is included. Microseparation techniques HPLC is a ubiquitous analytical tool in which the separation of analytes is based on their differential retention by a stationary phase. Typically, a stainless steel tube several millimeters in diameter is packed with particles coated with the stationary phase, and the sample to be separated is dissolved in a suitable solvent and passed through the tube by a pressure-driven flow. HPLC is useful for many analytical samples, but because of the inherent low efficiency resulting from the use of large-
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Report bore tubes, complex samples are often difficult to resolve. During the late 1970s and early 1980s, HPLC became the target of the miniatur ization trend in an attempt to gain higher column efficiencies and increased resolv ing power. Miniaturization would also de crease expenses associated with both the packing of large columns and the need for large volumes of solvent. The ability to sample from microenvironments with lim ited volumes was also an incentive for miniaturization. Thus the groundwork was laid for the development of micro LC and the use of small-bore glass or fusedsilica capillary columns instead of stain less steel tubes. The techniques that emerged from the use of capillaries are open-tubular liquid chromatography (OTLC) and microcol umn LC (2). OTLC has the higher effi ciency potential of the two techniques be cause, as its name implies, no packing material is used and therefore no eddy diffusion, which would add to solute band dispersion, exists. An OTLC column is prepared by coating the inner wall of a 3-50-μηι i.d. capillary column with the stationary phase. Alternatively, microcolumn LC in volves a capillary column packed with par ticles that are chemically modified to yield the stationary phase. There are two types of packed capillaries: partially packed (typically 40-80-μηι i.d.) and tightly packed (typically 40-200-pm i.d.). Partially packed columns can be made by packing a large-bore capillary with particles and pulling the capillary on a glass drawing machine to decrease the internal diame ter. The particles are then chemically modified to create the stationary phase. More often, however, a capillary is tightly packed by using slurry packing methods. In this case, a small-bore capillary is pres sure packed with chemically modified particles that are suspended in an organic solvent. Separations in open-tubular and packed microcolumns occur by the same retention process as in HPLC. A comple mentary separation mechanism is used in CZE (2,3). In general, CZE separation is carried out in a solvent-filled glass or fused-silica capillary across which a potentialas high as 40 kV is applied. The applied potential drives the separation 528 A
via two distinct phenomena. The first phenomenon, termed electroosmosis, produces a very strong flow toward the cathodic electrode caused by the interactions of positively charged sol vent components with the capillary inner wall (4). The second phenomenon, elec trophoresis, is the movement of charged species in an electric field. When charged analytes are introduced into the capillary, they undergo electrophoresis based roughly on their charge-to-size ratios and separate into discrete bands.
U¥~vis ahsorhance detection has been used extensively in the development of microseparation Sample introduction is carried out at the anodic end of the capillary in one of three ways: by applying the separation voltage for a few seconds to electromigrate a small plug of material, by raising the column entrance to induce gravity flow of the sample into the capillary, or by forcing the sample into the capillary via pressure. Generally, because of the electroosmoticflow,analytes migrate toward the cathodic end of the capillary whether they are positively or negatively charged. Electroosmotic flow has aflatflow pro file, unlike the laminar flow profile found in pressure-driven separation techniques, and this profile accounts in part for the very high separation efficiencies found in CZE. These high efficiencies are often reduced, however, by convection currents caused by resistive heating in the capil lary. Hjerten demonstrated increased sep arating power of zone electrophoresis in 1967 by rotating large-bore open capillar ies (5). The problem of joule heating has been addressed more recently through the miniaturization of electrophoresis techniques. The use of 200-500-μπι i.d. glass capil laries (6) and 200-μηι i.d. Teflon tubes (7)
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increased the surface area-to-volume ratio of the electrophoresis column, leading to improved heat dissipation across the liq uid — solid interface of the capillary. In the first use of capillaries with internal diame ters < 100 pm, efficiencies of > 400,000 theoretical plates were realized (8). Since that time, capillaries as small as 2-pm i.d. have been used (9), and CZE has become widely accepted as a high-efficiency microseparation technique. The development of microcolumn sep aration techniques allows quantitative analysis of volume-limited microenviron ments such as the contents of single cells (3,10,11). This analytical challenge re lies on the ability not only to sample ex tremely low volumes but also to detect analytes at low concentrations. The devel opment of sensitive detection schemes is therefore critical to the successful applica tion of microseparations. Early detection schemes for microsep arations were scaled-down versions of macroscale detectors. This approach was not very profitable, however, because macroscale detector designs were not easily adapted to low sample volumes. It was found that detectors were best made as a part of the microseparation column. UV—vis absorbance detection has been used extensively in the development of microseparation schemes, and it was thefirsttype of detector to be placed on a column. This was accomplished by remov ing the protective polyimide coating from the silica microcolumn to provide a detec tion window near its end. Because of the small-diameter capillaries used, however, the pathlength of these detection cells is very short and yields concentration detec tion limits of only 10"5 to 10~6 M. Also, problems involved in focusing and collect ing light onto and from the capillary have been limiting factors in the use of UV—vis absorbance for microseparations. Despite these drawbacks, this detection technique is still commonly used, because it is appli cable to a broad range of analytes. Ad vances toward more sensitive detection methods have been reported (12-17). Laser-induced fluorescence (LIF) is another detection scheme that has been applied to microseparations using the oncolumn format. In most cases, light from a laser is focused onto an on-column win dow and accounts for the increased sensi-
tivity of the technique. Although lasers offer a limited range of excitation wave lengths and samples need to be derivatized before analysis to make them fluorescently active, LIF detection is still attractive because low detection limits can be obtained. Attomole detection limits are routine, and zeptomole detection limits have been achieved by using a sheath flow chamber with optimized collection optics (18) and sensitive detectors with improved data collection methods (19). LIF detection has also been successfully applied to the analysis of single human erythrocytes using native fluorescence, derivatization, and indirect detection tech niques (20, 21). MS is another detection scheme com mon in macroscale separations that has been applied to microcolumn separations. This technique is attractive because of its general applicability and its ability to yield structural information. Coupling micro columns to MS is not as straightforward as coupling to optical detection methods because the on-column scheme cannot be used. Therefore, the technology for the routine use of MS in microseparations is still being developed. Much progress has been made, as outlined in a recent review article on CE/MS, which reports mass detection limits in the attomole range (22). Detection limits in the attomole range also have been reported for micro column LC with MS detection (23, 24). Electrochemical detection Electrochemical detection is successful in macroscale separations because of its ease of implementation; ability to selec tively detect and, in some cases, identify analytes; and high sensitivity. For these reasons it should also prove to be useful in microcolumn separations. In addition, electrochemical detection is a concentra tion-sensitive technique and therefore well suited for microseparations. As the vol ume of the microcolumn decreases, the mass detection limits should improve. The major drawback of electrochemical detection is the inherent selectivity of the technique, which generally limits analysis to easily oxidized or reduced species. However, this disadvantage can in some cases be circumvented, as will be dis cussed later. The three basic modes of electrochem-
ical detection are amperometry, conductimetry, and potentiometry. In the amperometric mode, compounds undergo oxidation or reduction reactions through the loss or gain, respectively, of electrons at the electrode surface. The electrical current arising from the electrons passed to or from the electrode is recorded and is proportional to the concentration of analyte present. Conductimetric and potentiometric methods measure the conductance or potential changes in the solution be tween two electrodes caused by the intro duction or removal of charged species. Amperometry is the most easily imple mented of the three modes of detection and is therefore the most commonly used. Coupling electrochemical detection cells to microcolumns was first attempted using the off-column format. However, the best detection schemes have involved on-
column detectors. The first on-column electrochemical detector for microcolumn LC was described by Manz and Simon in 1983 (25). An ion-selective electrode 1 μηι in diameter was inserted into the end of a 25-pm i.d. OTLC column to potentiometrically detect K* with detection limits in the picomole range. This same principle of on-column detection was used in 1984 by Knecht, Guthrie, and Jorgenson, who placed a 9-μπι diameter carbon fiber elec trode into the end of a 15-ujn i.d. OTLC column (26). Using amperometric detec tion, they achieved femtomole detection limits for ascorbic acid, catechol, and 4-methylcatechol. The development of electrochemical detection for CZE was not as straightfor ward as that for microcolumn LC because of the high potential fields used to effect the separation. In most cases, when an
Figure 1 . Electrochemical detection schemes for CZE. Analytical Chemistry, Vol. 66, No. 9, May 1, 1994 529 A
Report electrode was placed in the capillary, noise arising from the high voltage ap plied across the capillary interfered with detection. Despite this problem, two basic schemes have been developed that are routinely used for electrochemical detec tion with CZE. The first design was implemented in 1987 (27). A porous glass coupler, shown in Figure la, was used to decouple the electrode from the separation potential. The design involves a fracture near the
end of the capillary that is covered with porous glass to facilitate ion movement but not bulk flow. The separation voltage is therefore dropped across the capillary only up to the point of the fracture. Ap proximately 1 cm of capillary extends past the coupler and serves as the detection capillary. An electrode is manipulated into the detection capillary, which allows amperometry of analytes that are separated in the capillary and then electroosmotically pumped into the detection capillary.
Figure 2. Representative electropherograms of compounds separated using (a) a porous glass coupler design and (b) optimized end column detection. (a) Compounds detected: A, serotonin; B, norepinephrine; C, epinephrine; D, 1-dihydroxyphenylalanine; E, 5-hydroxyindoleaceticacid; F, homovanillic acid; G, dihydroxyphenylacetic acid; H, ascorbic acid. (Apparent volumes injected were 36 pL) (b) Compounds detected (apparent volumes injected in parentheses): A, dopamine (47 pL); B, isoproterenol (40 pL); C, catechol (27 pL). (Adapted from References 9 and 31.) 530 A
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An electropherogram of several com pounds, including catechols and ascorbic acid detected using the coupler design, is shown in Figure 2a. Detection limits as low as 0.5 amol have been reported for the separation of a standard solution of neu rotransmitters by using a 5-pm i.d. capil lary (9). The implementation of the coupler design permitted the first use of oncolumn electrochemical detection for CZE. However, the porous glass coupler is fragile and difficult to maintain. In 1992 O'Shea and co-workers (28) used a simi lar design with Nafion tubing instead of porous glass. This innovation provided an easier method for construction, resulting in a 100% success rate for larger capillar ies. A carbon fiber electrode was again used to detect analytes amperometrically with detection limits in the low-attomole range. A coupler using Teflon heat-shrunk tubing, which appears to be easier to con struct and to maintain than the original porous glass design, also has been re ported (29). The use of couplers made electrochemical detection in CZE possible by terminating the separation voltage, and therefore noise currents, before detection. The second approach to successful electrochemical detection has been to eliminate the need for couplers through the use of capillaries with small internal diameters (30). It appears that the poten tial field caused by the separation voltage decays very rapidly at the end of these capillaries, which allows electrodes to be manipulated up to the end of the column without the use of couplers (29,30). This technique has been demonstrated for the detection of several common neurotrans mitters with a 10-μπι diameter carbon fi ber electrode placed near the end of a cap illary, as shown in Figure lb (30). Detec tion limits as low as 56 amol have been reported for catechol; however, these re sults are tempered by difficulties in place ment of the electrode, which lead to low precision. Recently, a solution to the problem of end column electrode placement was de veloped (31). The end of a 5-pm i.d. capil lary is chemically etched to provide a coni cal entrance for electrode placement. A carbon fiber electrode is then easily in serted and held in the enlarged capillary exit, as shown in Figure lc. An electro-
pherogram of 10 ~ 6 M catechol detected with this method is shown in Figure 2b. Detection limits of 14-19 amol have been achieved for standard samples of com pounds commonly found in neurons. In addition to lower detection limits, more reproducible results have been obtained when compared with those obtained using the end column method. End column placement of an electrode has also been facilitated through the use of electrodes with diameters that are large relative to the internal diameter of the capillary (32). Detection limits compara ble to those obtained using end column detection have been achieved using microelectrodes, and run-to-run reproduc ibility using the large electrodes has been high. The noise arising from the separation potential is an important consideration in electrochemical detector design for CZE separations. In a recent study by Lu and Cassidy (29), several experiments were performed to determine the nature of this background noise and to provide insights into better detector designs. Examination of end column detection reveals that the separation voltage has a greater effect on noise when capillaries with large, rather than small, internal diameters are used. In addition, placement of the electrode is more critical for capillaries with larger internal diameters because of the slower decay of the electric field at the column end relative to small-bore capillaries. Cou pler designs have been compared with end column detection and are necessary when the internal diameter of the capillary is > 25 pm (29). These conclusions stress that careful electrode placement in end column detec tion configurations is important for im proving the detection limits of transition metals, inorganic ions, and catechols (33). Concentration detection limits of 5 χ 10 M have been reported for cate chol using a 25-pm i.d. capillary and a 10-pm diameter carbon fiber electrode. In addition, reported efficiencies for different electrode positions at the end of the capil lary are highly dependent on electrode placement. The best efficiencies are ob tained when the electrode is placed inside the capillary, apparently because convec tion currents and analyte diffusion outside the capillary are avoided.
Figure 3. Electropherogram of carbohydrates detected using PAD. Sample concentrations: 1 χ 10~4 M for inositol and 2 χ 10"4 M for others. Peak identification: 1, inositol; 2, sorbitol; 3, unknown; 4, maltose; 5, glucose; 6, rhamnose; 7, arabinose; 8, fructose and 9, xylose. (Adapted from Reference 38.)
The development of electrochemical detection for microseparations offers a highly sensitive method for the study of low-volume environments. However, the selectivity of the technique can limit the range of observable analytes. Much re search has been conducted to expand the range of analytes compatible with electro chemical detection. This research has flourished with the development of differ ent electrodes and detection schemes for CZE. E x p a n d i n g t h e r a n g e of detection
Because of its sensitivity, amperometry is the electrochemical detection mode most commonly used in microseparations; how ever, because the potential of the elec trode is held at one value, only those spe cies that are easily oxidized or reduced at that potential are detected. To extend the detectable range of analytes, voltammetry can be used. This technique involves ap plying a triangular potential waveform to the electrode to oxidize and reduce ana lytes that are electroactive in the potential range used. If the waveform is applied quickly enough, real-time voltammetric analysis of compounds eluting from a microseparation capillary can be obtained
(34). Fast-scan voltammetric analysis has the added advantage of analyte identifica tion, as different compounds yield differ ent characteristic voltammograms. In ad dition, co-eluting peaks can be resolved if the potential at which they are electroactive differs significantly. However, volt ammetry suffers from mass detection lim its approximately 100 times greater than those for amperometry. Another method that can be used to extend the range of analytes involves metal electrodes. Compounds not detect able at carbon fibers—such as alcohols, glycols, carbohydrates, sulfur compounds, amines, and amino acids—can interact with empty d-orbitals on the surface of a platinum or a gold electrode to form an electrochemically active species. A draw back that has limited the use of metal electrodes, though, is the tendency of electrochemically generated products to accumulate on the electrode surface. This process, termed electrode fouling or poi soning, results in the loss of the elec trode's ability to detect analytes. Fouling has been addressed through the use of pulsed amperometric detection (PAD), a well-established technique for HPLC (35). This technique involves rapid, continuous application of a three-step po-
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Report tential waveform to the electrode. The first step is used to detect the analytes; the second step, at a more positive potential, serves to electrochemically clean the surface of the electrode for the next detection pulse; and the third step is at a potential that reactivates the electrode for subsequent detection. The first use of PAD in a flowing stream with a microelectrode was demonstrated in the detection of carbohydrates at a gold electrode after separation by CZE (36). This study also included the use of pulsed voltammetric analysis to gain qualitative information. Further studies have used PAD for the detection of carbohydrates and metal ions (37, 38). A representative electropherogram of nine carbohydrates separated by CZE and detected with PAD is shown in Figure 3. The use of metal electrodes does not necessarily imply that PAD is required for successful detection. This was recently demonstrated for two types of metal electrodes. A copper electrode that was electrochemically pretreated to create a surface oxide layer was used to electrocatalytically detect carbohydrates separated with CZE (39). A single copper electrode was used for hundreds of separations during a two-week period with no deterioration in performance. A gold/mercury amalgam electrode was also used to selectively detect low levels of thiols through the catalytic oxidation of mercury—thiol species after CZE analysis (40). The use of metal electrodes has extended the range of observable analytes; however, each electrode discussed above is still generally applicable to a specific class of compounds. This is an advantage when selectivity is desired, but sometimes a broader class of compounds needs to be explored. Potentiometric or conductimetric analyses are alternative techniques that allow detection of more than one class of compounds. In the case of potentiometric analysis, ion-selective electrodes can be used to detect both inorganic and organic ions at the same time. These electrodes were used to detect several common neurotransmitters in addition to alkali metals (41, 42). With conductimetric analysis, the conductivity change is measured when an analyte band that has a mobility different from that of the solvent electro532 A
Figure 4. Chromatovoltammogram of a single nerve cell sampled from the land snail Helix aspersa. Peak identification: DA, dopamine; DHBA, 3,4-dihydroxybenzylamine; 5-HT, serotonin (two peaks result from oxidation of phenol and indole nitrogen at different potentials); TYR, tyrosine; TRP, tryptophan. (Adapted from Reference 50.)
Figure 5. Plot of norepinephrine versus epinephrine contained in individual bovine adrenomedullary cells. Cells containing an undetectable amount of norepinephrine were given a norepinephrine value of zero; the dashed line represents a catecholamine ratio of unity. (Adapted from Reference 51.)
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lyte enters the electrochemical detection region. As in potentiometry, this tech nique has been used to detect both metal and organic ions (43, 44). In addition, the recent development of suppressed con ductivity detection for CZE has resulted in more sensitive detection of analytes when compared with conventional conductimetric detection (45, 46). When the analyte of interest is not electroactive and cannot be directly de tected by electrochemical means, indirect detection can be used (47). This tech nique does not depend on oxidation or reduction of a specific class of com pounds. By adding an electrophore to the separation solvent, nonelectroactive spe cies can be detected as a dip in the back ground signal. Although this technique is useful for a broader range of solutes, there is a tradeoff with sensitivity. An alternative to the detection of non electroactive solutes by indirect detection is to use chemical derivatization to attach an electroactive group to the solute. This involves chemically reacting the sample prior to analysis with an electroactive tag that specifically derivatizes a target ana lyte. The most prevalent electroactive tag used is naphthalene-2,3-dicarboxaldehyde (NDA), which reacts with primary amines to form an electroactive compound. NDA derivatization methods have proven to be a valuable addition to electrochemical de tection techniques, as demonstrated by the analysis of amines in biological sam ples with CZE and OTLC (20, 48). Single-cell analysis One of the more challenging areas of bio logical analysis is the separation of the contents of single cells. Microcolumn LC and CZE can be used to sample volumes as low as nanoliters to femtoliters; there fore, the methodology exists to explore these ultrasmall environments. Because of its selectivity and sensitivity, electrochem ical detection is also well suited for study ing single cells. Several biologically inter esting species are electrochemically active, and the selectivity of electrochemi cal detection allows these species to be detected despite the complexity of biologi cal samples. The importance of performing singlecell analysis becomes apparent when the traditional means of determining cell con
tents and physiology are considered. Pool ing large cell populations for macroscale analysis is one technique that yields aver age values that are a composite of the at tributes of all cells present. This scheme limits the information gained about cellu lar function when a heterogeneous popu lation of cells is examined. For example, nowhere is the heterogeneity of cell types as complex as it is in the brain. Staining procedures can be used to determine cell contents, but the results are interpreted through visual inspection and are there fore subject to varying interpretations. Considering the problems faced in the investigation of individual cells, single-cell analysis via microseparation techniques becomes an attractive analytical tool. An example of single-cell analysis that led to new biological interpretations was the first whole-cell analysis of invertebrate neurons with OTLC (49). In this experi ment, an individual neuron from the land snail Helix aspersa was homogenized in a 500-nL microvial. The sample was centrifuged, and 1-100 nL of the supernatant was injected onto an OTLC column using a micropipette injector. Combined with this cell sampling and separation technol
ogy, fast-scan voltammetric detection was used to determine neurotransmitter and amino acid levels in three different neu rons (49, 50). Each of the three cells re sulted in characteristic chromatovoltammograms, one of which is shown in Figure 4. According to this analysis, two neurons of Helix aspersa contained both dopamine and serotonin, and another con tained neither neurotransmitter. All three neurons contained tyrosine and tryp tophan. These results refute Dale's hy pothesis that neurons contain only one type of neurotransmitter. In a separate report, 17 nonelectroactive amino acids in single nerve cells of Helix aspersa were determined amperometrically with OTLC after derivatization with NDA (48). The results from these experiments underline the importance of single-cell analysis. Single bovine adrenomedullary cells were recently analyzed using reversedphase microbore LC with amperometric detection (51). These cells are - 20 μπι in diameter and are 10-20 times smaller than invertebrate cells. This analysis rep resents an important step forward in single-cell sampling methodology. Nor epinephrine and epinephrine concen-
Figure 6. CZE analysis of a whole single nerve cell from the pond snail Planorbis corneus. Peak identification: A and Β are apparently dopamine (different retention times suggest storage in different vesicles); C, neutral species; D, uric acid; E, dihydroxyphenylacetic acid. (Adapted from Reference 55.) Analytical Chemistry, Vol. 66, No. 9, May 1, 1994 533 A
Report} trations were evaluated in individual cells ing (Figure 6). One of these peaks is abolturized and chemically or biologically to test the hypothesis that two distinct ished following treatment of the cell with modified electrodes should extend the types of adrenomedullary cells exist: one the vesicle-depleting drug reserpine. The range of analytes observed using electrocontaining mostly norepinephrine and the method shows promise as a means to chemical detection (58). If capillaries with other containing mostly epinephrine. The probe vesicular concentrations of dopasubmicrometer internal diameters beconcentrations of these components in 22 mine at the single-cell level. come available, amperometry will be well cells are shown in Figure 5. The results suited for detection. Electrodes with total indicate that most adrenal cells contain tip diameters of 400 nm have already been Future directions either norepinephrine or epinephrine; constructed and could be used with these In addition to aiding future analysis of sinfour cells, however, contained significant gle cells, the development of electrochem- capillaries (59). levels of both hormones. ical detection for microseparations will New microseparation methods should The work discussed above involved undoubtedly provide improvements in also benefit from the advantages of elecmanipulation of single cells in nanoliter detection limits and new applications. To trochemical detection. In our laboratories, microvials before the analysis. Single-cell increase sensitivity, techniques such as we have developed a continuous misampling can also be performed directly sinusoidal voltammetry can be used to croseparation technique based on CZE in a separation capillary for analysis by achieve picomolar detection limits in flow- (60). This has been accomplished via sepCZE with electrochemical detection (52, ing streams and are being applied to CZE aration in a channel structure that has an 53). The sample injection procedure for (56). More biological techniques, such as internal height and width of 48 pm and cytoplasmic samples involves chemically coupling CZE to microdialysis to monitor 2 cm, respectively. Sample introduction is etching the separation capillary at the in vivo release of amino acids (57), are on carried out using conventional CZE to sample introduction end to a fine tip of the horizon. The development of miniacontinuously sample material from mi6— lO-pm outer diameter (9, 53). The tip is inserted into the cell of interest, and a cytoplasmic sample is removed by applying the separation voltage for a few seconds to electroosmotically inject a small aliquot into the capillary. The capillary is then placed in a buffer solution, and separation is carried out. Cytoplasmic levels of the neurotransmitters dopamine and serotonin were determined amperometrically in two different neurons found in the pond snail Planorbis corneus (9, 54). The technology demonstrated in this study established a method for directly sampling a single-cell cytoplasm into a capillary electrophoretic system. CZE in 25-pm i.d. capillaries was also used to sample single whole neurons in Planorbis corneus (53). In this experiment, electroosmotic flow was used to inject a cell into an etched capillary, and subsequent injection of nonphysiological buffer was used to lyse the cell membrane. This methodology has recently been used to test the hypothesis that neurotransmitters exist in two vesicular compartments in a nerve cell (55). Apparently, one compartment is for immediate use in neurotransmission and the other is for longer term storage. Changing the waiting period for Figure 7. Experimental setup for continuous electrophoresis in narrow cell lysis following injection into the end channels. of the capillary results in a dramatic (a) Sample is placed in reservoir 1, and sampling capillary is attached to a stepper motor that change in the electropherogram. Short moves it across the entrance of the channel. The channel is suspended across buffer reservoirs 2 and 3. (b) Representation of the separation process in the channel for three analytes with lyse times result in two peaks for dopadifferent mobilities. Left: the analytes first being deposited into the channel. Middle: the migration mine that can be attributed to the different paths after the capillary has moved to the middle of the channel. Right: the capillary at the end of resistances of the two vesicle types to lysthe channel entrance, where sample deposition is typically halted. (Adapted from Reference 60.) 534 A
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croenvironments. The end of the CZE capillary is inserted into the entrance of the channel and is slowly moved across its width to allow continuous deposition of sampled material. Material migrates in the channel in straight paths and separates along the 5-cm length of the channel. The principles of this new separation mode are illustrated in Figure 7. Although LIF detection with fiber-optic arrays to deliver excitation light and col lect fluorescence has been used to demon
strate the separation method, we are cur rently implementing an electrochemical detection scheme for continuous channel separations. The detector consists of 100 gold microelectrodes chemically depos ited across the channel plate at its exit. Each electrode is individually addressed to provide a response based on the posi tion of an eluting analyte across the chan nel exit width. Figure 8 shows a sche matic of the channel with a micrograph of the electrode array detector. The electro-
chemical detection array should provide improved detection limits and will allow further miniaturization of the electro phoresis channel internal height. In conclusion, electrochemical detec tion has emerged as one of the more pow erful detection methods for microseparations because of its selectivity, sensitivity, and ability to detect low levels of many biologically relevant molecules without prior derivatization. The future of electro chemical detection appearsjo be bright, as evidenced by recent developments in the construction of new electrodes and the implementation of different detection schemes. In addition, the use of microseparations to investigate single cells has demonstrated the applicability of this technique in biochemical analyses. We acknowledge our co-workers whose efforts are referenced in this Report. We thank NSF, NIH, and the Office of Naval Research for financial support. We also thank Dave Lilienfeld, Garry Bordonaro, and the NSF-funded National Nanofabrication Facility at Cornell University for assistance in developing the microelectrode array.
Figure 8. Schematic of electrode array detector for continuous electrophoresis in narrow channels. Beige rectangular portion represents the top channel plate (quartz, 2.5 χ 5.0 χ 0.23 cm). Electroactive region is composed of 100 individually addressed gold microelectrodes (each 95 pm wide; 5-pm interelectrode spacing). The electrodes are fabricated directly onto the bottom channel plate (quartz, 7.5 χ 7.5 χ 0.23 cm) by standard microlithographic techniques. An individual address to each electrode is accomplished through the termination at the appropriate contact'pad. The inset micrograph shows a 10-electrode portion of the array. The white horizontal portions are electroactive regions; the black portions are quartz substrate.
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